Recombinant Human Interleukin Research Articles

IL-8 and PI3K pathway influence the susceptibility of TRAIL-sensitive colorectal cancer cells to TRAIL-induced cell death.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an apoptosis inducer that exhibits an ideal therapeutic safety profile with less adverse effects than conventional chemotherapy. However, the occurrence of TRAIL resistance has been reported in various cancers including colorectal cancer (CRC). Substantial efforts have been channelled towards managing TRAIL resistance including identifying molecular targets. Interleukins (ILs) have been recently shown to play critical roles in modulating TRAIL sensitivity in cancer cells. This study investigated the roles of two ILs, IL-8 and IL⍺, in TRAIL resistance in CRC. TRAIL-resistant HT-29 and TRAIL-sensitive HCT 116 cells, were treated with human recombinant IL-8 and IL-1⍺. The results indicated that treatment with IL-8 (2.5 ng/mL) significantly protected TRAIL-sensitive HCT 116 cells from TRAIL-induced cell death (p < 0.05). However, IL-1⍺ did not play a role in modulating CRC cells' responses to TRAIL. Data from RT-qPCR and Western blotting revealed the molecular regulations of IL-8 on TRAIL decoy receptor genes (OPG) and autophagy-related genes (BECN1 and LC3B) expression. The activation of the phosphoinositide 3-kinase (PI3K) pathway was shown to counteract TRAIL-induced cell death. By inhibiting its activation with wortmannin, the protective role of IL-8 against TRAIL treatment was reversed, suggesting the involvement of the PI3K pathway. Collectively, findings from this study identified the role of IL-8 and PI3K in modulating CRC cells' sensitivity to TRAIL. Further validation of these two potential molecular targets is warranted to overcome TRAIL resistance in CRC.

Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy

BackgroundPediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient...

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome

Post-COVID syndrome develops in 10–20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

Xuanbi Yuyang Decoction Ameliorates DSS-Induced Colitis by Inhibiting Pyroptosis via Blocking of IL-17 Pathway Activation.

Ulcerative colitis (UC), a highly relapsing non-specific disease, is difficult to cure completely. The investigation aims to determine the protective effect and potential action mechanism of Xuanbi yuyang decoction (XBD) on UC. The chemical composition of XBD was determined through non-targeted metabolomics analysis. Subsequently, experimental mice were orally given 3% DSS for 6 days, followed by XBD treatment (0.3 mL, 0.4 mL). In vitro, the human colon epithelial cells were co-treated with DSS and medicated serum. The therapeutic effects of XBD on UC were evaluated in vivo and vitro. The mechanisms of XBD against UC were determined by detecting hallmarks related to pyroptosis and Interleukin (IL)-17 pathways using Western blot and ELISA. The recombinant human interleukin 17A (rhIL17A) and was applied for further verifying the effect of XBD on IL-17 pathway in UC cells. XBD supplementation restored DSS-induced weight loss, colon shortening and tissue damage, and reduced DAI. Moreover, XBD enhanced viability, repaired the intestinal mucosal barrier of colitis, decreased pro-inflammatory cytokines levels, and inhibited pyroptosis. Additionally, DSS increased the expression of IL-17 pathway was and cytokines (IL-17A, IL-6), which were blocked by XBD treatment. The rhIL17A treatment attenuated protective effect against DSS-induced colitis and could also enhance pyroptosis. XBD has a favorable protective effect against DSS-induced colitis through restraining pyroptosis via inhibition of IL-17 signaling pathway activation, suggesting XBD may be a new and effective treatment therapy for UC.

Transiently boosting Vγ9+Vδ2+ γδ T cells early in Mtb coinfection of SIV-infected juvenile macaques does not improve Mtb host resistance.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children, despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4-8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, (n=5; i.v.) at 3- and 17- days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group (n=5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy eight weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.

Arecoline promotes fibroblast activation and M2-macrophage polarization by up-regulating the expression of IL-4

ObjectiveTo determine the biological effects of arecoline on oral submucous fibrosis (OSF). DesignThe differential genes between OSF tissue and normal oral tissue were collected form GSE64216 dataset, analyzed by Gene Expression Omnibus (GEO) database. Real-time PCR and immunohistochemistry were used to analyze the expression of IL-4 gene and protein in oral tissue. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of exocrine IL-4 protein in human oral fibroblasts (HOF) pre-treated by arecoline. Cell Counting Kit-8 (CCK-8) and transwell assays were used to analyze the proliferation and migration of HOF cells, respectively. After IL-4 was knocked down by short hairpin (sh) plasmid, the proliferation and migration of HOF cells were detected. Flow cytometry was used to analyze the proportion of M2-macrophages. Real-time PCR and immunohistochemistry were used to verify the expression of biomarker proteins of macrophages in OSF tissues. ResultsThe expression of IL-4 gene and protein were both up-regulated in OSF tissue. Arecoline could enhance the expression of IL-4 gene and exocrine protein in HOF cells, and promote the proliferation and migration of HOF cells. While knockdown of IL-4 could inhibit arecoline-induced proliferation and migration in HOF cells. The results of flow cytometry showed that recombinant human IL-4 (rhIL-4) protein could increase the proportion of M2-macrophages. Similarly, the results of real-time PCR and immunohistochemistry showed the expression of ARG1 (Biomarker proteins of M2-macrophage) was up-regulated in OSF tissues. ConclusionArecoline promotes activation of fibroblasts and polarization of M2-macrophages by up-regulating the expression of IL-4.

Chrysin Enhances Anti-cancer Activity of Jurkat T Cell and NK-92 Cells Against Human Breast Cancer Cell Lines.

Chrysin, a naturally occurring flavonoid in plant and bee products, demonstrates notable biological activities, including anti-cancer effects. These properties are partially attributed to its capability to activate immune cells. This study focused on exploring the immunomodulatory potential of chrysin on NK-92 and Jurkat-T cells targeting breast cancer cells (BCC). Chrysin leads to activation of NK-92 and T cells facilitated by the addition of human recombinant IL-2 and PHA-M. The anti-cancer efficacy of chrysin on these immune cells was evaluated in a co-culture setup with EGF-stimulated MCF-7 and MDA-MB-231 cells. Findings revealed that chrysin notably increased the cytotoxicity of NK-92 and T cells towards MCF-7 and MDA-MB-231 cells, with the most significant impact observed on MCF-7 cells (20%). The activation of NK-92 cells, marked by increased IFN-γ production and CD56 expression, correlated with enhanced secretion of cytokines. Additionally, the activation of these cells against BCC was linked with elevated levels of granzyme-B, TNF-α, and nitric oxide (NO). Similarly, the cytotoxic activation of Jurkat-T cells against BCC was characterized by increased production of granzyme-B, IL-2, and IFN-γ. Consequently, these results support the hypothesis that chrysin significantly contributes to the activation and functional enhancement of NK-92 and T-cells against two distinct BCC lines.

Hepatic artery infusion pump (HAIP) therapy in combination with targeted delivery of IL-12 for patients with metastatic colorectal cancer or intrahepatic cholangiocarcinoma: a phase II trial protocol.

Treatment of advanced liver tumors remains challenging. Although immune checkpoint inhibition has revolutionized treatment for many cancers, responses in colorectal liver metastases and biliary tract cancers remain suboptimal. Investigation into additional immunomodulatory therapies for these cancers is needed. Interleukin-12 (IL-12) is a pro-inflammatory cytokine with robust anti-tumor activity, but systemic adverse effects largely terminated therapeutic development of recombinant human IL-12 (rhIL-12). PDS01ADC is a novel human monoclonal antibody (NHS76) conjugated to two IL-12 heterodimers with established safety in phase I trials. The NHS76 antibody specifically targets histone/DNA complexes which are accessible only in regions of cell death and this antibody has been shown to accumulate locally in tumors. Patients with unresectable metastatic colorectal cancer (mCRC) or unresectable intrahepatic cholangiocarcinoma (ICC) will receive synchronization of subcutaneous PDS01ADC with floxuridine delivered via a hepatic artery infusion pump (HAIP). The primary outcome measured in this study will be overall response rate as measured by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Secondary outcomes measured in this study will include hepatic and non-hepatic progression-free survival (PFS), overall survival, and safety of PDS01ADC combination therapy with HAIP. Poor clinical response of these liver tumors to immunotherapy is likely due to various factors, including poor immune infiltrate into the tumor and immunosuppression by the tumor microenvironment. By exploiting the tumor cell death induced by HAIP locoregional therapy in combination with systemic chemotherapy, PDS01ADC is poised to modulate the tumor immune microenvironment to improve outcomes for patients undergoing HAIP therapy. ClinicalTrials.gov (ID NCT05286814 version 2023-10-18); https://clinicaltrials.gov/study/NCT05286814?term=NCT05286814&rank=1.

LPS-related muscle loss is associated with the alteration of Bacteroidetes abundance, systemic inflammation, and mitochondrial morphology in a weaned piglet model.

We previously demonstrated that lipopolysaccharide (LPS) injection-induced immune stress could impair muscle growth in weaned piglets, but the precise mechanisms behind this remain elusive. Here, we found that chronic immune stress induced by LPS resulted in a significant reduction of 36.86% in the total muscle mass of piglets at 5 d post-treatment compared with the control group. At 1 d, prior to muscle mass loss, multiple alterations were noted in response to LPS treatment. These included a reduction in the abundance of Bacteroidetes, an increase in serum concentrations of pro-inflammatory cytokines, compromised mitochondrial morphology, and an upregulation in the expression of dynamin-related protein 1 (Drp1), a critical protein involved in mitochondrial fission. We highlight a strong negative correlation between Bacteroidetes abundance and the levels of serum pro-inflammatory cytokines, corroborated by in vivo intervention strategies in the musculature of both pig and mouse models. Mechanistically, the effects of Bacteroidetes on inflammation and muscle mass loss may involve the signaling pathway of the tauro-β-muricholic acid-fibroblast growth factor 15. Furthermore, the induction of overexpression of inflammatory cytokines, achieved without LPS treatment through oral administration of recombinant human IL-6 (rhIL-6), led to increased levels of circulating cytokines, subsequently causing a decrease in muscle mass. Notably, pre-treatment with Mdivi-1, an inhibitor of Drp-1, markedly attenuated the LPS-induced elevation in reactive oxygen species levels and rescued the associated decline in muscle mass. Collectively, these data indicate that LPS-induced muscle mass loss was linked to the reduction of Bacteroidetes abundance, increased inflammation, and the disruption of mitochondrial morphology. These insights offer promising avenues for the identification of potential therapeutic targets aimed at mitigating muscle mass loss.

Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Therapeutic potential of the topical recombinant human interleukin-1 receptor antagonist in guinea pigs with allergic rhinitis

BackgroundRecombinant human Interleukin receptor antagonist (rhIL-Ra) can bind to the IL-1 receptor on the cell membrane and reversibly blocks the proinflammatory signaling pathway. However, its effect on allergic rhinitis (AR) and the underlying mechanism remains unknown. This study aims to investigate the efficacy of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on AR guinea pigs.MethodsGuinea pigs were systemically sensitized by intraperitoneal injection and topical intranasal instillation with ovalbumin within 21 days. Animals administrated with saline served as the normal control. The AR animals were randomly divided into the model group and distinct concentrations of rhIL-1Ra and budesonide treatment groups. IL-1β and ovalbumin specific IgE levels were detected by ELISA kits. Nasal mucosa tissues were stained with hematoxylin & eosin (HE) for histological examination.ResultsIt was found that the numbers of sneezing and nose rubbing were remarkably reduced in rhIL-1Ra and budesonide-treated guinea pigs. Besides, rhIL-1Ra distinctly alleviated IgE levels in serum and IL-1β levels in nasal mucus, together with decreased exfoliation of epithelial cells, eosinophilic infiltration, tissue edema and vascular dilatation.ConclusionsrhIL-1Ra is effective in AR guinea pigs and may provide a novel potential choice for AR treatments.

MB2033, an anti-PD-L1 × IL-2 variant fusion protein, demonstrates robust anti-tumor efficacy with minimal peripheral toxicity

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rβγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rβγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

Abstract LB118: HM16390, a novel long-acting IL-2 analog with fine-tuned binding affinities to IL-2 receptor subunits for favorable safety profile, exhibits potent tumor killing effect in the various tumor syngeneic models

Abstract Although recombinant human IL-2 (rhIL-2, aldesleukin) produced approximately a 15% objective response rate in patients with renal cell carcinoma (RCC) and metastatic melanoma, its IL-2Rα (CD25)-biased binding and short half-life (t1/2 in human: 13-85 min) require a high dose and frequent dosing interval, leading to systemic toxicity such as vascular leak syndrome (VLS) and cytokine release syndrome (CRS). To overcome this limitation, various research groups are developing engineered IL-2 muteins with abolished CD25 binding affinity. While such IL-2 muteins may reduce CD25-mediated toxicity, there is a risk of a lopsided immune response. Furthermore, concomitantly reduced affinity to IL-2Rβ (CD122) may cause insufficient anti-tumor activity. We are developing HM16390, a long-acting IL-2 analog with a significantly increased binding affinity to CD122 for potent tumor killing effect. Simultaneously, optimal binding property to CD25 was introduced for a suppression of uncontrolled systemic immune response via peripheral Treg expansion. From these novel sequence modifications, HM16390 exhibited approximately 14-fold potent activity in human NK and CD8+ T cells compared to rhIL-2 and comparable activity with rhIL-2 in human Tregs (EC50 of STAT5 phosphorylation) without undesired release of cytokines associated with CRS. In B16F10 mouse model, HM16390 has a longer-lasting PK property (t1/2: 14.2~26.0 hr) after single subcutaneous (SC) administration. CD8+ T cells were significantly increased in peripheral blood along with a transient increase of peripheral Tregs, suggesting that HM16390 not only has a significant effect on expansion of CD8+ T cells but can simultaneously modulate exaggerated peripheral immune response. It is also noteworthy that, unlike in the peripheral, Tregs were drastically reduced in the tumor immune microenvironment, and CD8+ T cells were significantly increased. Next, in orthotopic model of mouse RCC (RENCA-luc cell implantation), across treatment groups, 40%(n=4/10) to 80%(n=8/10) of animals achieved complete tumor regression in dose-dependent manner after once weekly SC injection of HM16390 for 4 weeks. Furthermore, tumor nodules were not observed in lung and kidney after intravenous (IV) rechallenge of RENCA cells in the mice cured by HM16390 for at least 4 months after drug discontinuation. This indicates a long-term immunological memory response of generated memory cells by treatment of HM16390. In conclusion, HM16390 demonstrates potent and durable anti-tumor activity in various murine tumor models via its enhanced CD122 binding affinity, and incorporated optimal CD25 binding affinity may alleviate systemic toxicity by immunomodulatory role of peripheral Tregs. Citation Format: Sol-Bi Shin, Jinyoung Kim, Jaehyuk Choi, Seongju Jeong, Yunjae Kim, Jooyun Byun, Sungmin Bae, Daejin Kim, In Young Choi. HM16390, a novel long-acting IL-2 analog with fine-tuned binding affinities to IL-2 receptor subunits for favorable safety profile, exhibits potent tumor killing effect in the various tumor syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB118.

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics

BackgroundDespite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge...

Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism

BackgroundNatural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index.MethodsA semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor βγ (IL-2Rβγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rβγ complex.ResultsPhenotypic screening in mouse identifies SAR444336 (SAR’336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR’336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR’336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation.ConclusionSAR’336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.

Chemerin promotes invasion of oral squamous cell carcinoma by stimulating IL-6 and TNF-α production via STAT3 activation.

Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro. Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays. Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies. Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling.

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6744.

Abstract 2837: Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha

Abstract Dogs share physiological similarities with humans, positioning them as valuable models for studying the efficacy and safety of drugs and their underlying mechanisms of action. The JAK-STAT pathway, a highly conserved signaling pathway across species, including humans and dogs, presents an opportunity to investigate the therapeutic potential of cytokines that utilize this pathway. In this study, we aimed to evaluate the functional activity of three canine cytokines on canine cells. To assess stimulation of the JAK/STAT pathway, we used a flow cytometry assay that measures phospho-STAT in activated canine or human lymphocytes in whole blood specimens. Testing of commercially available human and canine IL-6 proteins revealed that canine lymphocytes respond poorly to most canine IL-6 products, whereas all recombinant human IL-6 products stimulated STAT phosphorylation in canine lymphocytes, despite sharing low sequence similarity with canine IL-6. In human T cells, both canine IL-6 and human IL-6 stimulated STAT phosphorylation, demonstrating biological activity from the canine IL-6 products. Several JAK inhibitors inhibited cytokine-induced STAT phosphorylation in both canine and human CD4 T-cells, consistent with JAK inhibition, but targeting the IL-6 receptor with tocilizumab only blocked IL-6-induced STAT phosphorylation in human but not canine CD4 T-cells in whole blood. These findings demonstrate the potential of our assay system to assess cross-species cytokine reactivity, determine if JAK/STAT inhibitors used clinically might be useful in treating inflammatory conditions in dogs, and identify novel canine-specific inhibitors. Additionally, we explored the induction of IL-8 by human and canine IL-1 alpha in IL-1 alpha sensitive human cells. Human IL-1 alpha failed to stimulate a canine IL-1 alpha sensitive cell line, suggesting species-specific differences in IL-1 alpha activity. Furthermore, we discovered that canine IL-7 and human IL-7 are species-specific. In summary, this proof-of-concept study highlights the capability to assess the activation of STATs and pathway inhibition by JAK inhibitors in canine whole blood using flow cytometry. We confirmed that human IL-6 stimulates JAK/STAT signaling in canine CD4 T-cells. Our results also revealed significant differences in species cross-reactivity of human IL-6, IL-7, and IL-1 alpha, underscoring the importance of species-specific investigations. Leveraging the similarities and differences between canine and human cytokines targeting the JAK-STAT pathway can enhance drug development efforts and contribute to the advancement of safer and more effective therapies for both humans and pet dogs with similar diseases. This study was funded by NCI Contract No. 75N91019D00024. Citation Format: King Leung Fung, Melissa L. Breiner, James B. Mitchell, Ralph E. Parchment. Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2837.